Protocol Online logo
Top : Forum Archives: : Immunology and Histology

antibody inactivation of peptide - ELISA troubleshooting (Aug/18/2006 )

I've heard that one can inactivate a peptide with an antibody...does anyone know how?
I am working on an ELISA where I coat the plate with mAb, block nonspecific binding, incubate with samples and recombinant protein standards, block again, then apply a biotinylated Ab, apply HRP-strep, then develop with a TMB solution. My standards don't show up at all, but my samples do.
So I'm beginning to suspect that the mAb and the biotinylated Ab might be interfering with each other in the recombinant standards, or that the biotinylated Ab can't bind the recombinant protein. If I preincubate a sample of the recombinant protein with mAb, then run a western probing with the biotinylated Ab...and they interfere, I shouldn't see a band...right? How would I do this?


I suspect your standard to be degraded.

you should run a gel, stain with coomassie, or silver and check if you can see your recombinant protein.